Endometrial cancer is the most common primary gynecologic malignancy in the United States, with over 39,000 new cases diagnosed each year. Most women in the US present with endometrioid endometrial carcinoma. This histologic subtype carries a favorable prognosis, with an overall 5-year survival that approaches 90%. Despite the fact that most patients are cured with surgery, recurrent endometrioid adenocarcinoma is a significant cause of mortality. Adjuvant radiation therapy, given in an attempt to eliminate any remaining tumor cells, is often a part of the treatment plan for women who are at increased risk for recurrent disease. In women with early stage (I and II) endometrial carcinoma, the use and benefits of adjuvant therapy are controversial. Radiation therapy carries a significant risk for complications. Furthermore, the costs for radiation therapy are considerable. In a large multicenter randomized trial of adjuvant therapy for intermediate risk endometrioid adenocarcinoma, it was demonstrated that pelvic radiation significantly decreased recurrences but brought about only a small, non-statistically significant improvement in overall survival. There is a need to find a balance between the increased cost and morbidity that comes with adjuvant radiation therapy, and the reduction in the recurrences for women with intermediate risk disease. A molecular marker to identify those patients who are at very low-risk for recurrence could help avoid unnecessary adjuvant therapy and by doing so, lessen the morbidity and expense associated with treating endometrial cancer. Conversely, a marker for high-risk disease could help target the use of adjuvant therapy. Furthermore, the ability to recognize those women at higher risk for aggressive or deadly disease prior to hysterectomy could have a positive effect on how this group of women is cared for. The long-term goal for the marker studies proposed here is to combine molecular stratification and conventional risk assessment in prospective randomized trials. In this application we propose to develop a new method to analyze methylation of rDNA sequences in clinical tumor specimens. We previously demonstrated that rDNA methylation was an independent prognostic marker in patients with endometrioid endometrial adenocarcinoma. We will develop 1) Pyrosequencing TM methods to assess rDNA methylation in tumor DNAs, 2) apply the rDNA Pyrosequencing TM methylation analyses to clinical specimens (paraffin-embedded, formalin-fixed hysterectomy and pre-hysterectomy, biopsies) and 3) in the R33 phase of the application, we will use these methods to assess the prognostic and diagnostic significance of Pyrosequencing TM rDNA methylation in a consecutive series of women with endometrioid adenocarcinoma. These studies are designed to determine whether tumor rDNA methylation could serve as a prognostic and diagnostic marker in both pre-hysterectomy and hysterectomy specimens from women with endometrial cancer, rDNA methylation analyses could ultimately be used to help guide the treatment of endometrial cancer patients